The invention relates generally to the field of gas phase ion fragmentation techniques, and more precisely to electron capture dissociation (ECD) which is used to fragment gas-phase analyte ions such as large biopolymer ions in order to obtain structural information via mass spectrometry.
A gas-phase ion fragmentation technique frequently used in the field of mass spectrometry is the collision-induced dissociation (CID), sometimes also called collisionally activated dissociation (CAD). Molecular ions are usually accelerated by an electrical potential to high kinetic energy and then allowed to collide with quasi-stationary neutral molecules of a background gas, such as helium, nitrogen or argon which are largely chemically inert in order to prevent chemical reactions from occurring. In the collision, some of the kinetic energy is converted into internal energy which results in bond breakage and the fragmentation of the molecular ion into smaller fragments, at least some of which carry unbalanced charges. These charged fragment ions can then be analyzed by a mass spectrometer, such as a linear or three-dimensional quadrupole mass analyzer, linear or orthogonal accelerated time-of-flight analyzer, ion cyclotron resonance analyzer and the like.
Electron-capture dissociation, initially described by Roman Zubarev, Neil Kelleher, and Fred McLafferty (Zubarev et al. (1998); “Electron capture dissociation of multiply charged protein cations. A nonergodic process”; J. Am. Chem. Soc.; 120 (13): 3265-3266), on the other hand, is a gas-phase ion fragmentation method which taps the energy reservoir of a recombination reaction between cations and free electrons. ECD involves the mixing of low energy electrons with gas phase ions which, according to recent developments, can be trapped in a suitable trapping device, such as 3D (Paul type) ion trap, 2D linear ion trap and the like. An example of such a trap arrangement is disclosed, for example, in U.S. Pat. No. 7,755,034 to Ding.
An ECD reaction normally involves a multiply protonated molecule M interacting with a free electron to form an odd-electron ion:[M+nH]n++e−→[[M+nH](n-1)+]*→fragments.
Adding an electron to an incomplete molecular orbital of the reactant cation releases binding energy which, if sufficient to exceed a dissociation threshold, causes the fragmentation of the electron acceptor ion.
ECD produces significantly different types of fragment ions, primarily of the c and z type, than aforementioned CID which primarily yields the b and y type. CID introduces internal vibrational energy in the cation in an ergodic process generally affecting the weakest bonds and thus causing loss of post-translational modifications (PTM) such as phosphorylation and O-glycosylation during fragmentation. In ECD, on the other hand, these PTMs are largely retained in the fragments. Consequently, in ECD unique fragments can be observed which are largely complementary to CID fragments thereby allowing a more detailed structural elucidation of the reactant cation. However, low fragmentation efficiencies and other experimental difficulties, in particular the problem of simultaneously confining ions with high masses and light electrons (the mass of an electron is about 1,836 times smaller than that of a proton), posed a hindrance hitherto for the utility of ECD. A further challenge is to provide electrons with sufficiently low kinetic energy as to allow electron capture reactions to occur.
Another gas-phase ion fragmentation technique tapping the energy reservoir of a recombination reaction is called electron-transfer dissociation (ETD). Similar to electron-capture dissociation, ETD induces fragmentation of cations of interest, such as peptides or proteins, by an electron transfer from a suitable reagent anion, both reactants normally being confined in an ion trap. The scientific potential of this process using polyaromatic reagent anions was first realized by Donald Hunt, Joshua Coon, John Syka and Jarrod Marto (Syka et al. (2004); “Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry”; Proc. Natl. Acad. Sci. U.S.A.; 101 (26): 9528-9533; see also U.S. Pat. No. 7,534,622 to Hunt et al.).
In contrast to ECD, ETD does not use free electrons but employs anions, preferably radical polyaromatic anions of anthracene or fluoranthene, as electron donors in a charge transfer reaction:[M+nH]n++A−→[[M+nH](n-1)+]*→fragments
where A− is the anion. Just like ECD, the ETD fragmentation technique is considered beneficial as it cleaves randomly along the peptide backbone of the electron acceptor cation in a non-ergodic process, yielding fragments of the c and z type, while side chains and modifications such as phosphorylations are left intact. Therefore, ETD, as much as ECD, is complementary to CID and is thought to be advantageous for the fragmentation of longer peptides or even entire proteins raising its value for top-down proteomics. One reason why ETD is nowadays in more widespread use than ECD is that the masses of the reactant cations and anions do not diverge as much as the masses of reactant cations and electrons making it easier to simultaneously confine them in an ion trap, for instance. On the other hand, one difficulty with ETD is that the electron transfer reactions compete with other reaction types such as proton transfer, ion attachment and the like, resulting in different individual branching ratios and ETD yields that depend on the pair of reagents used. Such competition of reaction pathways does not exist with ECD.
Since the first application of ECD in an ion cyclotron resonance cell the technique associated therewith was further advanced. Glish et al. (US 2004/0245448 A1), for example, describe a mass spectrometer capable of performing ECD that comprises a first mass analyzer, a magnetic trap downstream of the first mass analyzer, a second mass analyzer downstream of the magnetic trap, and an electron source positioned such that electrons are supplied to the magnetic trap. Whitehouse et al. (U.S. Pat. No. 6,919,562 B1 and U.S. Pat. No. 7,049,584 B1) disclose an apparatus that enables the interaction of low energy electrons with sample ions to facilitate ECD within multipole ion guide structures. Voinov et al. (Rapid Commun. Mass Spectrom., 2008, 22(19), 3087-3088) report on ECD performed in a linear, radio frequency free, hybrid electrostatic/magnetostatic cell without the aid of a cooling gas.